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Losses of metabolically incorporated selenium in common digestion procedures for biological material
~alanto. vol. 29, pp. 1025to 1028,1982
0039-9140/82/l11025~04$03.00/0
Printed in Great Britain.All rights reserved
Copyright 0 1982 Pergamon Press Ltd
LOSSES OF METABOLICALLY INCORPORATED SELENIUM IN COMMON DIGESTION PROCEDURES FOR BIOLOGICAL MATERIAL H. J.
ROBBERECHT,* R. E. VAN GRIEKEN,* P. A. VAN DEN BOSCH,~ H. DEELSTRA?
D. Departments
and
VANDEN BERGHE$
of Chemistry,* Pharmacy? and Medicine,§ University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Wilrijk, Belgium (Received
14 January 1982. Accepted
30 April 1982)
Summary-Two common procedures for wet destruction of biological materials for subsequent determination of selenium have been investigated. Rat organs and biological fluids were endogenously labelled with ‘%e to monitor losses during the procedures. Addition of nitric and perchloric acids with gradual heating up to 210” seemed to be the best method: at this temperature the labelled selenium was still recovered quantitatively, and the destruction was fast and efficient.
the discovery that selenium is essential to human health, the need for information about the concentration of this element in human tissues has become more urgent. For the determination of trace elements in biological samples a pretreatment is needed which will destroy all organic matter but retain the element of interest. As some selenium species are known to be volatile at higher temperatures, it is necessary to investigate possible losses at every step of the determination procedure. For the drying step, Behne and Matamba’ found no differences were caused in the selenium concentrations in blood serum by drying at 90”. Fourie and Peisach2-4 studied the loss of selenium metabolically incorporated in zoological specimens and found no significant losses caused by drying at up to 105”. Iyengar et aL5 studied the retention of radioactive selenium incorporated into biological tissues when different drying procedures were used and proved that, with heating at up to 120”, the loss was less than 5%. This evidence indicates that the drying of biological material is not a critical step. For the decomposition step, Hall and Gupta6 found selenium losses from 10% up to 60% during wet ashing with nitric and perchloric acids at temperatures from 150” up to 200”. We therefore first tested a digestion procedure with 110” as the highest temperature. A predigestion step at a lower temperature was included in this procedure, as suggested by Olson,’ with the aim of hydrolysing easily oxidizable biological material. The temperature was then gradually increased in the presence of a sufficient amount of nitric acid to prevent charring of the organic material and consequent evolution of H,Se from the digestion mixture.‘-” Olson’ stated that since some organoselenium compounds can resist acid attack by perchloric
Since
acid up to 200”, the destruction has to proceed at above this temperature for at least 15 min. After re-
covery experiments with the (CH&Se+ ion, Olson er al.” extended the heating period to 30 min. Hall and Gupta6 warned against using temperatures above 230”. Therefore in the second digestion procedure tested, the final temperature was set at 210”. In a recent recommendation for the determination of selenium in biological materialsI sulphuric acid is added, but only after complete oxidation of the biological matrix, since it is especially useful for driving off the remaining nitric and perchloric acids, both of which interfere in the recommended fluorimetric determination procedure. However, since sulphuric acid enhances the risk of charring the biological matrix 11V138’4 it was not added in the two digestion procedures used in this study. All the published studies on the recovery of selenium during wet destruction have made use of spikes, sometimes containing radioactive tracers, which were added to the samples on the assumption that the matrix selenium and the spike, mostly added in the inorganic form, would behave similarly during the matrix decomposition. 13*15,16This approach is questionable, however, since the concentration ranges of the trace elements in biological tissues vary over several orders of magnitude and especially since it has not been confirmed that the inorganic form and the organically bound selenium behave in the same way in the decomposition procedures. A better appraisal is obtained by the analysis of standard reference materials, which have been analysed for selenium by a non-destructive technique, and which resemble the various types of biological samples as closely as possible. In another approach, possible losses can be studied during digestion of tissues in which radioactive selenium has been biologically incorporated. 1’ In the present investigation the radioactive ‘%e isotope was injected intraperitoneally into rats in
1025
H.J.
1026
ROBBERECHT et al.
order to incorporate this element into the body tissues during normal metabolic functioning and to study the recovery of the selenium in its naturally occurring form, when the two different wet digestion procedures were applied.
EXPERIMENTAL
Reagents and apparatus All reagents were of analytical grade. In all experiments ‘%e (tli2 = 120 days; E, = 265 keV) was used. Sodium selenite with initial specific activity 5.9 nCi per pg of Se, and sodium selenate with initial specific activity 6.3 &i per ng of Se, were obtained from the Radiochemical Centre, Amersham, England. The various organs and biological fluids were decomposed in a 20-position Tecator automatic digestion apparatus (Hogan%, Sweden), including a No. 1005 heating unit and a No. 1008 control unit. The total height of the digestion tubes and specially constructed condensers was 55cm. In the first set of experiments, the gamma-emitting nuclide was counted on a Ge(Li) detector with a 4096-channel analyser. In the second experiment a Berthold automatic gamma samplechanger unit LB MAG 312 (Wildbad, Germany) was used for quantitatively analysing the radioactivity. The detector consists of a 3 x 3 in. NaI(T1) well-type scintillation crystal with a well diameter of 22 mm and is rather insensitive to small variations in counting geometry. Procedure Two adult Wistar rats with average body weight of 250 g were injected intraperitoneally with 4 bug of selenate (25 PCi) and 4.25 pg of selenite (25 $i). After injection the rats were placed in metabolism cages for collection of urine and faeces. Standard food was supplied and they received water ad fibitum. Every 24 hr. urine and faeces were collected and after 9 days the animals were killed. Samples of whole blood, brain, heart, kidney, liver and spleen were removed. The initiat radioactivities of 75Se were measured in all samples. The biological tissues were taken for destruction and the residual activity in the empty vials was checked. The digestion procedure was based on the comprehensive literature evaluation of Verlinden.” Concentrated nitric acid (5 ml per g of sample) was added to react with the organs overnight at room temperature; subsequently the solution was heated at 70” for 23 hr and then at 100” for 20 hr. The condensers were then removed, the volume was reduced to a few ml and 2 ml of perchloric acid were added. The mixtures were heated for 1 hr at 107” with the condensers replaced. After cooling and addition of 1 ml of concentrated hydrochloric acid the mixture was heated for 5 min at 100” to reduce all selenium to the quadrivalent state, so that it could be determined by common analytical procedures. The radioactivity of the solutions obtained after the destruction of organic matter was measured with the same geometry in the Ge(Li) detector. In a second experiment the rats were kept alive for 30 days after injection of 33 pg of ‘sSe (80 PCi). Half of the selenium was added as seienite, the other half as selenate. This higher amount of selenium is still only one thirtieth of the reported minimum lethal dose of selenate or selenite injected into rats,‘9,20 so it is improbable that the metabolic incorporation was occurring under pathological conditions. The rats were killed, and the initial radioactivity was measured in the whole blood, brain, heart, kidney, liver, lung, muscle, spleen and thyroid. Urine and faeces were collected during the 30 days and were also included in this tracer experiment. The digestion scheme finally adopted, in the litera<.,n.P,.l following ., .“_. recommendations ture,““~y”“~‘J“4”0‘” mvolved addmon of concentrated
nitric acid (10 ml per g of sample) to the organs and predigestion overnight at room temperature; heating at 75” for 24 hr and then at 120” for 6 hr until the solution volume was reduced to 4 ml; addition of 5 ml of perchloric acid (70%) and heating (up to 215”) for 30 min; cooling and reduction of selenium to the quadrivalent state by heating for 5 min with concentrated hydrochloric acid. A standard solution containing 0.66 ng of ‘sSe (0.34 pg of selenium as selenite and 0.32 pg as selenate) was also taken through the entire decomposition procedure. The radioactivities before and after destruction were measured in the NaI(TI) detector.
RESULTS
AND
DISCUSSION
Table 1 summarizes the results for the retention of the radioactive isotope after decomposition at the lower temperature (< 1 lo”) of heart, liver, spleen, kidneys, brain, blood, urine and faeces of the two rats following 9 days of metabolic incorporation. For the biological fluids, the recovery is nearly complete and the reproducibility satisfactory. For some of the tissues the results are apparently too high and the reproducibility is somewhat poor. This is due to some uncertainties in the counting geometry, that could not be avoided during the counting of the whole organs and faeces because, in view of the low subtoxic quantities of selenium used, the gamma-activities were low, necessitating measurement close to the Ge(Li) detector. Still, it seems that, in the low-temperature decomposition process, the selenium is retained virtually quantitatively in the final solution. However, some lipid material remained and the faeces were not completely destroyed in this procedure. Verlindeni8 also found low selenium concentrations for blood and serum by AAS after this decomposition step. In the second experiment, the incorporation of a higher amount of selenium took place over a longer period, and muscle and lungs were also investigated because selenium is known to affect muscle dys-
Table 1. Recovery of 75Se, metabolically incorporated for 9 days, after decomposition at 1 loRecoveries,*
Samples Heart Liver Spleen Kidney Brain Blood Urine (after 1 day) (after 6 days) (after 8 days) Faeces (after 1 day) (after 6 days) (after 8 days) Overall mean
“,;
90, -95,126 95.111 112,95 117,107 95,95 93,97 94,102 102, ~~ 103, 103, ~ 114,102 + 10
*Individual values, Ge(Li) spectrometry,
measured by for two rats.
Losses of metabolically incorporated
selenium
1027
Table 2. Specific activity of “Se metabolically incorporated for 1 month, in different organs and biological fluids, and its recovery after decomposition by the extended wet-acid procedure at high temperature (210”) Specific ‘?Se activity, cpmlg
Samples Heart Liver Spleen Kidney Brain Blood Urine (1 (2 (1 Thyroid Muscle Lung Standard
week) weeks) month)
‘?Se solution
Recovery,* %
18.0 x lo3 38.4 x lo3 28.5 x lo3 58.0 x lo3 7.8 x lo3 26.1 x lo3 18.4 x lo3 13.0 x lo3 8.5 x lo3 15.1 x lo3 10.0 x lo3 17.1 x lOA 6.5 x 10s Overall mean:
Concentration found, M/g wet weight
lOl,-, 91 95, 107,102 99,102 110,96 89, 82, ~ 102, 84, 109,90 111,96 97,100 97 + 8
0.33 1.23 0.42 1.21 0.14 0.36
0.35 0.11 0.32
*Individual values, measured by NaI(TI) and Ge(Li) spectrometry, for one rat. trophy, and selenium accumulation in the lungs has been reported. 22,23 The samples were also measured with a well-type NaI(T1) detector that is less sensitive to geometry effects. Before measurement of the initial radioactivity on the Ge(Li) detector, the organs and fluids were allowed to stand for 24 hr in l&20 ml of concentrated nitric acid, so that a more comparable geometry was obtained before and after the digestion. Table 2 summarizes the results for the recovery of incorporated 75Se after the extended wet destruction with nitric and perchloric acids, and with heating to 210”. The highest specific activities were found in kidney, liver, spleen and blood, while brain showed the lowest activity. These findings agree quite well with the literature data on the distribution of subtoxic amounts of 75Se in the tissues of rats.24 Liver and kidney seem to be the target organs in exposure of rats to selenium,23~z5 whereas in man the lungs appear to accumulate the selenium.22,23 Harr et a[.25 stated that the liver-to-kidney selenium ratio is dependent on the level of selenium supply, so after addition of higher doses, higher liver-to-kidney ratios are found. This could be explained by the fact that the conversion of selenite into volatile selenium compounds takes place largely in the hver,26 and that these can be exhaled by the rats.27 Another way of eliminating high amounts of selenium is in the urine, in which the trimethylselenonium ion is the major metabolite excreted:28p31 hence, the kidneys will also show higher selenium concentrations. From Table 2 it can be concluded that even at the high decomposition temperature of 210” the recovery of “Se is almost complete for the different organs. The clear solutions obtained after digestion also point to a complete destruction of fatty materials. Hence this method seems excellent for determination of selenium in biological samples. Only thyroid and some
respectively,
urine samples showed a somewhat lower recovery. No explanation can yet be offered for the lower recovery from thyroid tissue. For urine, losses of selenium after wet or dry ashing are widely reported in the literature. Schwarz32 recommended a closed system for accurate analysis, since serious losses of the element, as much as 75-84%, were observed during open wet digestion. In a dry ashing method for urine, Roquebert and Truhaut33 found serious losses. Probably incomplete digestion of the volatile selenonium ion is the major reason for these losses. Geahchan and Chambon31 however, found no difference in selenium recovery when they compared destruction of urine in a closed system (Teflon Parr bomb), with an open digestion method (using a nitric-perchloric acid mixture) similar to the procedures presented above.
CONCLUSION
Selenium-75 was injected intraperitoneally to incorporate the element into the various organs of rats in a natural physiological form. The highest specific activity was found in kidney, liver, spleen and blood, while brain accumulated the least activity. Heating with nitric-perchloric acid mixture up to 210” appeared to be a most convenient method for decomposing the different organs and biological fluids: the matrix was completely destroyed, and no significant losses of the incorporated selenium could be observed (except for thyroid and some urine samples). The decomposition procedure is rather complex and long, but can easily be performed in an automatic digestion apparatus. The resulting solutions can be used for selenium determination by, for example, hydride-generation atomic-absorption spectrometry18 or energy-dispersive X-ray fluorescence analysis after selective reduction and preconcentration on activated charcoal.34
1028
H. J. ROBBERECHT et al.
Acknowledgements-H.R. and P.V.D.B. acknowledge the financial support of the Belgian Ministry of Health, via a research project on the impact of selenium in the Belgian environment. REFERENCES 1. D. Behne and P. A. Matamba, 2. Anal. Chem., 1975, 274, 195. 2. H. 0. Fourie and M. Peisach, Radiochem. Radioanal. Lett., 1976, 26, 277. 3. Idem, S. Afr. J. Sci., 1976, 72, 349. 4. Idem, Analyst, 1977, 102, 193. 5. G. V. Iyengar, K. Kasperek and L. E. Feinendegen, Sci. Total Environ., 1978, 10, 1. 6.
Talanta,1974,21,859. 15. J. Piick. J. Hoste and J. Gillis, Proc. Intern. Symp. Microchem., p. 48. Pergamon Press, Oxford, 1960. 16. Analvtical Methods Committee, Analvst. 1965, 90. 515. 17. D. 6 Reamer and C. Veillon, Anal.~ Chem., 1981, 53, 1192.
18. M. Verlinden, Ph.D. Thesis, University of Antwerp (UIA), 1981. 19. K. W. Franke and A. L. Moxon, J. Pharmacol. Exp. Ther., 1936, 58, 454. 20. I. S. Palmer and 0. E. Olson, Biochem. Biophys. Res. Commun., 1979, 90, 1379. 21. 0. E. Clinton, Analyst, 1977, 102, 187. 22. M. Yukawa, M. Suzuki-Yasumoto, K. Amano and M. Terai, Arch. Intern. Med., 1979, 139, 824. 23. C. .I. Diskin, C. F. Tomasso, J. C. Alper, M. L. Glaser and S. E. Fliegel, Arch. Intern. Med., 1979, 139, 824. 24. L. L. Hopkins Jr., A. L. Pope and C. A. Baumann, J. Nutr., 1966, 88, 61. 25. J. R. Harr. J. H. Exon. P. H. Weswig and P. D. Whanger, Clin. Toxicol., 1973, 6, 487. 26. A. Shrift. Bat. Rev.. 1958. 24. 550. 27. H. E. Ganther, 0. k. Leiander and C. A. Baumann, J. Nutr., 1966, 88, 155. 28. J. L. Byard, Arch. Biochem. Biophys. 1969, 130, 555. 29. I. S. Palmer, D. D. Fisher, A. W. Halverson and 0. E. Olson, Biochim. Biophys. Acta, 1969, 177, 336. 30. I. S. Palmer, R. P. Gunsalus, A. W. Halverson and 0. E. Olson, ibid., 1970, 208, 260. and P. Chambon, C/in. Chem., 1980, 26, 31. A. Geahchan 1272. 32. K. Schwarz, Selenium in Biomedicine, p. 112. AVI Publishing Co., Westport, 1967. 33. J. P. Roquebert and R. Truhaut, Bull. Sot. Pharm. Bordeaux, 1962, 101, 143. 34. H. J. Robberecht and R. E. Van Grieken, Anal. Chim. Acta (submitted).
0039-9140/82/l11025~04$03.00/0
Printed in Great Britain.All rights reserved
Copyright 0 1982 Pergamon Press Ltd
LOSSES OF METABOLICALLY INCORPORATED SELENIUM IN COMMON DIGESTION PROCEDURES FOR BIOLOGICAL MATERIAL H. J.
ROBBERECHT,* R. E. VAN GRIEKEN,* P. A. VAN DEN BOSCH,~ H. DEELSTRA?
D. Departments
and
VANDEN BERGHE$
of Chemistry,* Pharmacy? and Medicine,§ University of Antwerp (U.I.A.), Universiteitsplein 1, B-2610 Wilrijk, Belgium (Received
14 January 1982. Accepted
30 April 1982)
Summary-Two common procedures for wet destruction of biological materials for subsequent determination of selenium have been investigated. Rat organs and biological fluids were endogenously labelled with ‘%e to monitor losses during the procedures. Addition of nitric and perchloric acids with gradual heating up to 210” seemed to be the best method: at this temperature the labelled selenium was still recovered quantitatively, and the destruction was fast and efficient.
the discovery that selenium is essential to human health, the need for information about the concentration of this element in human tissues has become more urgent. For the determination of trace elements in biological samples a pretreatment is needed which will destroy all organic matter but retain the element of interest. As some selenium species are known to be volatile at higher temperatures, it is necessary to investigate possible losses at every step of the determination procedure. For the drying step, Behne and Matamba’ found no differences were caused in the selenium concentrations in blood serum by drying at 90”. Fourie and Peisach2-4 studied the loss of selenium metabolically incorporated in zoological specimens and found no significant losses caused by drying at up to 105”. Iyengar et aL5 studied the retention of radioactive selenium incorporated into biological tissues when different drying procedures were used and proved that, with heating at up to 120”, the loss was less than 5%. This evidence indicates that the drying of biological material is not a critical step. For the decomposition step, Hall and Gupta6 found selenium losses from 10% up to 60% during wet ashing with nitric and perchloric acids at temperatures from 150” up to 200”. We therefore first tested a digestion procedure with 110” as the highest temperature. A predigestion step at a lower temperature was included in this procedure, as suggested by Olson,’ with the aim of hydrolysing easily oxidizable biological material. The temperature was then gradually increased in the presence of a sufficient amount of nitric acid to prevent charring of the organic material and consequent evolution of H,Se from the digestion mixture.‘-” Olson’ stated that since some organoselenium compounds can resist acid attack by perchloric
Since
acid up to 200”, the destruction has to proceed at above this temperature for at least 15 min. After re-
covery experiments with the (CH&Se+ ion, Olson er al.” extended the heating period to 30 min. Hall and Gupta6 warned against using temperatures above 230”. Therefore in the second digestion procedure tested, the final temperature was set at 210”. In a recent recommendation for the determination of selenium in biological materialsI sulphuric acid is added, but only after complete oxidation of the biological matrix, since it is especially useful for driving off the remaining nitric and perchloric acids, both of which interfere in the recommended fluorimetric determination procedure. However, since sulphuric acid enhances the risk of charring the biological matrix 11V138’4 it was not added in the two digestion procedures used in this study. All the published studies on the recovery of selenium during wet destruction have made use of spikes, sometimes containing radioactive tracers, which were added to the samples on the assumption that the matrix selenium and the spike, mostly added in the inorganic form, would behave similarly during the matrix decomposition. 13*15,16This approach is questionable, however, since the concentration ranges of the trace elements in biological tissues vary over several orders of magnitude and especially since it has not been confirmed that the inorganic form and the organically bound selenium behave in the same way in the decomposition procedures. A better appraisal is obtained by the analysis of standard reference materials, which have been analysed for selenium by a non-destructive technique, and which resemble the various types of biological samples as closely as possible. In another approach, possible losses can be studied during digestion of tissues in which radioactive selenium has been biologically incorporated. 1’ In the present investigation the radioactive ‘%e isotope was injected intraperitoneally into rats in
1025
H.J.
1026
ROBBERECHT et al.
order to incorporate this element into the body tissues during normal metabolic functioning and to study the recovery of the selenium in its naturally occurring form, when the two different wet digestion procedures were applied.
EXPERIMENTAL
Reagents and apparatus All reagents were of analytical grade. In all experiments ‘%e (tli2 = 120 days; E, = 265 keV) was used. Sodium selenite with initial specific activity 5.9 nCi per pg of Se, and sodium selenate with initial specific activity 6.3 &i per ng of Se, were obtained from the Radiochemical Centre, Amersham, England. The various organs and biological fluids were decomposed in a 20-position Tecator automatic digestion apparatus (Hogan%, Sweden), including a No. 1005 heating unit and a No. 1008 control unit. The total height of the digestion tubes and specially constructed condensers was 55cm. In the first set of experiments, the gamma-emitting nuclide was counted on a Ge(Li) detector with a 4096-channel analyser. In the second experiment a Berthold automatic gamma samplechanger unit LB MAG 312 (Wildbad, Germany) was used for quantitatively analysing the radioactivity. The detector consists of a 3 x 3 in. NaI(T1) well-type scintillation crystal with a well diameter of 22 mm and is rather insensitive to small variations in counting geometry. Procedure Two adult Wistar rats with average body weight of 250 g were injected intraperitoneally with 4 bug of selenate (25 PCi) and 4.25 pg of selenite (25 $i). After injection the rats were placed in metabolism cages for collection of urine and faeces. Standard food was supplied and they received water ad fibitum. Every 24 hr. urine and faeces were collected and after 9 days the animals were killed. Samples of whole blood, brain, heart, kidney, liver and spleen were removed. The initiat radioactivities of 75Se were measured in all samples. The biological tissues were taken for destruction and the residual activity in the empty vials was checked. The digestion procedure was based on the comprehensive literature evaluation of Verlinden.” Concentrated nitric acid (5 ml per g of sample) was added to react with the organs overnight at room temperature; subsequently the solution was heated at 70” for 23 hr and then at 100” for 20 hr. The condensers were then removed, the volume was reduced to a few ml and 2 ml of perchloric acid were added. The mixtures were heated for 1 hr at 107” with the condensers replaced. After cooling and addition of 1 ml of concentrated hydrochloric acid the mixture was heated for 5 min at 100” to reduce all selenium to the quadrivalent state, so that it could be determined by common analytical procedures. The radioactivity of the solutions obtained after the destruction of organic matter was measured with the same geometry in the Ge(Li) detector. In a second experiment the rats were kept alive for 30 days after injection of 33 pg of ‘sSe (80 PCi). Half of the selenium was added as seienite, the other half as selenate. This higher amount of selenium is still only one thirtieth of the reported minimum lethal dose of selenate or selenite injected into rats,‘9,20 so it is improbable that the metabolic incorporation was occurring under pathological conditions. The rats were killed, and the initial radioactivity was measured in the whole blood, brain, heart, kidney, liver, lung, muscle, spleen and thyroid. Urine and faeces were collected during the 30 days and were also included in this tracer experiment. The digestion scheme finally adopted, in the litera<.,n.P,.l following ., .“_. recommendations ture,““~y”“~‘J“4”0‘” mvolved addmon of concentrated
nitric acid (10 ml per g of sample) to the organs and predigestion overnight at room temperature; heating at 75” for 24 hr and then at 120” for 6 hr until the solution volume was reduced to 4 ml; addition of 5 ml of perchloric acid (70%) and heating (up to 215”) for 30 min; cooling and reduction of selenium to the quadrivalent state by heating for 5 min with concentrated hydrochloric acid. A standard solution containing 0.66 ng of ‘sSe (0.34 pg of selenium as selenite and 0.32 pg as selenate) was also taken through the entire decomposition procedure. The radioactivities before and after destruction were measured in the NaI(TI) detector.
RESULTS
AND
DISCUSSION
Table 1 summarizes the results for the retention of the radioactive isotope after decomposition at the lower temperature (< 1 lo”) of heart, liver, spleen, kidneys, brain, blood, urine and faeces of the two rats following 9 days of metabolic incorporation. For the biological fluids, the recovery is nearly complete and the reproducibility satisfactory. For some of the tissues the results are apparently too high and the reproducibility is somewhat poor. This is due to some uncertainties in the counting geometry, that could not be avoided during the counting of the whole organs and faeces because, in view of the low subtoxic quantities of selenium used, the gamma-activities were low, necessitating measurement close to the Ge(Li) detector. Still, it seems that, in the low-temperature decomposition process, the selenium is retained virtually quantitatively in the final solution. However, some lipid material remained and the faeces were not completely destroyed in this procedure. Verlindeni8 also found low selenium concentrations for blood and serum by AAS after this decomposition step. In the second experiment, the incorporation of a higher amount of selenium took place over a longer period, and muscle and lungs were also investigated because selenium is known to affect muscle dys-
Table 1. Recovery of 75Se, metabolically incorporated for 9 days, after decomposition at 1 loRecoveries,*
Samples Heart Liver Spleen Kidney Brain Blood Urine (after 1 day) (after 6 days) (after 8 days) Faeces (after 1 day) (after 6 days) (after 8 days) Overall mean
“,;
90, -95,126 95.111 112,95 117,107 95,95 93,97 94,102 102, ~~ 103, 103, ~ 114,102 + 10
*Individual values, Ge(Li) spectrometry,
measured by for two rats.
Losses of metabolically incorporated
selenium
1027
Table 2. Specific activity of “Se metabolically incorporated for 1 month, in different organs and biological fluids, and its recovery after decomposition by the extended wet-acid procedure at high temperature (210”) Specific ‘?Se activity, cpmlg
Samples Heart Liver Spleen Kidney Brain Blood Urine (1 (2 (1 Thyroid Muscle Lung Standard
week) weeks) month)
‘?Se solution
Recovery,* %
18.0 x lo3 38.4 x lo3 28.5 x lo3 58.0 x lo3 7.8 x lo3 26.1 x lo3 18.4 x lo3 13.0 x lo3 8.5 x lo3 15.1 x lo3 10.0 x lo3 17.1 x lOA 6.5 x 10s Overall mean:
Concentration found, M/g wet weight
lOl,-, 91 95, 107,102 99,102 110,96 89, 82, ~ 102, 84, 109,90 111,96 97,100 97 + 8
0.33 1.23 0.42 1.21 0.14 0.36
0.35 0.11 0.32
*Individual values, measured by NaI(TI) and Ge(Li) spectrometry, for one rat. trophy, and selenium accumulation in the lungs has been reported. 22,23 The samples were also measured with a well-type NaI(T1) detector that is less sensitive to geometry effects. Before measurement of the initial radioactivity on the Ge(Li) detector, the organs and fluids were allowed to stand for 24 hr in l&20 ml of concentrated nitric acid, so that a more comparable geometry was obtained before and after the digestion. Table 2 summarizes the results for the recovery of incorporated 75Se after the extended wet destruction with nitric and perchloric acids, and with heating to 210”. The highest specific activities were found in kidney, liver, spleen and blood, while brain showed the lowest activity. These findings agree quite well with the literature data on the distribution of subtoxic amounts of 75Se in the tissues of rats.24 Liver and kidney seem to be the target organs in exposure of rats to selenium,23~z5 whereas in man the lungs appear to accumulate the selenium.22,23 Harr et a[.25 stated that the liver-to-kidney selenium ratio is dependent on the level of selenium supply, so after addition of higher doses, higher liver-to-kidney ratios are found. This could be explained by the fact that the conversion of selenite into volatile selenium compounds takes place largely in the hver,26 and that these can be exhaled by the rats.27 Another way of eliminating high amounts of selenium is in the urine, in which the trimethylselenonium ion is the major metabolite excreted:28p31 hence, the kidneys will also show higher selenium concentrations. From Table 2 it can be concluded that even at the high decomposition temperature of 210” the recovery of “Se is almost complete for the different organs. The clear solutions obtained after digestion also point to a complete destruction of fatty materials. Hence this method seems excellent for determination of selenium in biological samples. Only thyroid and some
respectively,
urine samples showed a somewhat lower recovery. No explanation can yet be offered for the lower recovery from thyroid tissue. For urine, losses of selenium after wet or dry ashing are widely reported in the literature. Schwarz32 recommended a closed system for accurate analysis, since serious losses of the element, as much as 75-84%, were observed during open wet digestion. In a dry ashing method for urine, Roquebert and Truhaut33 found serious losses. Probably incomplete digestion of the volatile selenonium ion is the major reason for these losses. Geahchan and Chambon31 however, found no difference in selenium recovery when they compared destruction of urine in a closed system (Teflon Parr bomb), with an open digestion method (using a nitric-perchloric acid mixture) similar to the procedures presented above.
CONCLUSION
Selenium-75 was injected intraperitoneally to incorporate the element into the various organs of rats in a natural physiological form. The highest specific activity was found in kidney, liver, spleen and blood, while brain accumulated the least activity. Heating with nitric-perchloric acid mixture up to 210” appeared to be a most convenient method for decomposing the different organs and biological fluids: the matrix was completely destroyed, and no significant losses of the incorporated selenium could be observed (except for thyroid and some urine samples). The decomposition procedure is rather complex and long, but can easily be performed in an automatic digestion apparatus. The resulting solutions can be used for selenium determination by, for example, hydride-generation atomic-absorption spectrometry18 or energy-dispersive X-ray fluorescence analysis after selective reduction and preconcentration on activated charcoal.34
1028
H. J. ROBBERECHT et al.
Acknowledgements-H.R. and P.V.D.B. acknowledge the financial support of the Belgian Ministry of Health, via a research project on the impact of selenium in the Belgian environment. REFERENCES 1. D. Behne and P. A. Matamba, 2. Anal. Chem., 1975, 274, 195. 2. H. 0. Fourie and M. Peisach, Radiochem. Radioanal. Lett., 1976, 26, 277. 3. Idem, S. Afr. J. Sci., 1976, 72, 349. 4. Idem, Analyst, 1977, 102, 193. 5. G. V. Iyengar, K. Kasperek and L. E. Feinendegen, Sci. Total Environ., 1978, 10, 1. 6.
Talanta,1974,21,859. 15. J. Piick. J. Hoste and J. Gillis, Proc. Intern. Symp. Microchem., p. 48. Pergamon Press, Oxford, 1960. 16. Analvtical Methods Committee, Analvst. 1965, 90. 515. 17. D. 6 Reamer and C. Veillon, Anal.~ Chem., 1981, 53, 1192.
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